人端粒酶逆转录酶真核表达质粒转染人成纤维细胞的体外培养及生物学特性研究

CULTURE OF HUMAN FIBROBLASTS TRANSFECTED BY HUMAN TELOMERASE REVERSE TRANSCRIPTASE EUCARYOTIC EXPRESSION PLASMID pGRN145 AND THEIR BIOLOGICAL CHARACTERISTICS IN VITRO

目的 分析人端粒酶逆转录酶 (h TERT)真核表达质粒 p GRN14 5转染人成纤维细胞的生物学特性。方法 体外分离培养小儿包皮成纤维细胞 ,脂质体法将构建的 p GRN14 5转染人成纤维细胞 ,经潮霉素 B筛选 ,扩增培养阳性克隆。 RT- PCR法、TRAP- PCR法分析细胞端粒酶活性。流式细胞术检测细胞增殖周期及细胞凋亡率 ,对转染后细胞行染色体核型分析及免疫组织化学法检测胶原分泌能力。结果 转染后培养的人成纤维细胞形态与转染前相比无明显改变 ,转染细胞稳定地表达端粒酶活性 ,流式细胞术分析转染前后成纤维细胞的增殖周期无明显改变 ,细胞凋亡率较转染前降低。经 p GRN14 5转染的人成纤维细胞保持正常的二倍体核型 ,具有正常分泌 、 型胶原的能力。结论 p GRN14 5转染的人成纤维细胞稳定地表达端粒酶活性 ,细胞寿命明显延长。

Objective To analysis the biological characteristics of human fibroblasts transfected by human telomerase reverse transcriptase (hTERT) eucaryotic expression plasmid pGRN145. Methods Fibroblasts from children's foreskin were isolated and cultured in vitro , and the fibroblasts were transfected by pGRN145 with LipofectAMINE PLUS TM Reagent. After strict screening of hygromycin B, the positive clones were subcultured. The telomerase activity was detected by RT PCR and TRAP PCR technique. The cell generation cycle and apoptosis rate were detected by flow cytometry to investigate the proliferative characteristics after transfection, and the chromosome karyotype of transformed cells was analyzed. The collagen secreted by transformed cells was detected by immunohistochemical staining. Results The morphological properties of fibroblasts did not change obviously after transfection. There were telomerase activity in transfected fibroblasts, while it could not be detected in pre transfection fibroblasts. The cell generation cycle had no obvious changes between pre transfection and post transfection. However, the apoptosis rate of transfected fibroblasts were decreased compared with that of pre transfection. The fibroblasts transfected by pGRN145 maintained the normal diploid karyotype, as well as the cells could normally secret type Ⅰand Ⅲ collagen. Conclusion The human fibroblasts transfected by pGRN145 has telomerase activity with prolonged life span of culture, which preliminarily proves the availability of establishing standard seeding cell lines of tissue engineering by hTERT plasmid transfection techniques.